It is well known that mosquitoes and other dipterans serve as vectors for diseases such as malaria, encephalitis, sleeping sickness, yellow fever, and dengue fever. In most instances, disease causing organisms not only successfully penetrate the well protected, hard and tanned exoskeleton of the insect vector, but also invade their immune system. Our long term goals are a) to unravel the structure and mechanism of formation of crosslinks stabilizing the insect cuticle; b) to examine the biochemistry of the enzymes involved in sclerotization, wound healing, and melanization; and c) to study the developmental and regulatory role of these enzymes at the macromolecular level. A better understanding of these processes can eventually lead to the development of new and target oriented insect control measures. The specific aims are: a) to study the enzymology of cuticular sclerotization; b) to continue model sclerotization studies and crosslink analysis in dipteran cuticle; and c) to examine the prophenoloxidase activation and its regulation. To achieve these goals, the following studies will be carried out: Quinone isomerase and quinone methide isomerase - two new enzymes involved in sclerotization of insect cuticle will be purified and characterized from S. bullata, D. melanogaster, and Aedes aegypti. The possible formation of a metabolon by phenoloxidase - quinone isomerase and quinone methide isomerase will be evaluated. The metabolism of dehydro N-beta- alanyldopamine will be examined by biosynthetic and oxidative studies. Polyclonal antibodies to prophenoloxidase and quinone isomerase will be obtained and used to determine the intracellular localization and to examine the synthesis, processing, and regulation of these enzymes. Chemical and enzymatic model sclerotization studies will be continued. Adducts of quinones and quinone methides with small and macromolecular compounds will be obtained by synthetic and degradative routes and their structure will be established using UV, IR, and NMR spectroscopic techniques. The role of phenoloxidase, quinone isomerase, and other proteins involved in sclerotization and melanization of insect cuticle will be evaluated. The mechanism(s) of activation of prophenoloxidase system in the hemolymph will be determined.